Department of Biochemistry, National Institute of Nutrition (Indian Council of Medical Research), Jamai-Osmania, Hyderabad-500 007, Andhra Pradesh, India. E-mail: email@example.com
Objective: Scavenger receptor class BI (SR-BI), authentic high-density lipoprotein (HDL) receptors expressed in liver, are known to play an important role in HDL-cholesterol (C) metabolism and reverse cholesterol transport. Interestingly, obese rats of WNIN/Ob strain have abnormally elevated levels of serum HDL-C compared with their lean counterparts. Based on the well-established role of SR-B1 in HDL-C metabolism, it was hypothesized that these obese rats may have an underexpression of hepatic SR-B1 receptors. In view of the significant role of vitamin A in energy expenditure and obesity, we also tested whether vitamin A supplementation can correct abnormal HDL-C metabolism.
Research Methods and Procedures: To test this hypothesis, 7-month-old male lean and obese rats of WNIN/Ob strain were divided into two groups; each group was subdivided into two subgroups consisting of six lean and six obese rats and received diets containing either 2.6 or 129 mg vitamin A/kg diet for 2 months.
Results: At the end, obese rats receiving normal levels of vitamin A diet showed high serum HDL-C and lower hepatic SR-BI expression levels compared with lean counterparts. Furthermore, chronic dietary vitamin A supplementation resulted in overexpression of hepatic SR-BI receptors (protein and gene) with concomitant reduction in serum HDL-C levels in obese rats.
Discussion: Thus, our observations highlight the role of vitamin A in reverse cholesterol transport through up-regulation of hepatic SR-BI receptors and, thereby, HDL-C homeostasis in obese rats of WNIN/Ob strain.
Reverse cholesterol transport (RCT)1 is one of the major mechanisms by which excess cholesterol from extrahepatic tissues is removed, transported to liver, and secreted into bile (1, 2). High-density lipoprotein (HDL) is the main component of the RCT process (3). Epidemiological studies have clearly established the inverse relationship between high HDL-cholesterol (C) and coronary heart disease (4). However, the discovery of unique receptors, namely scavenger receptor class BI (SR-BI) involved in the regulation of plasma HDL-C levels through RCT, cardioprotection, steroidogenesis, and reproduction, has aroused alot of scientific interest. SR-BI is an 82-kDa protein with 509 amino acids and a member of the CD-36 superfamily of proteins. Furthermore, rodent SR-BI shares extensive homology with human SR-BI (5). These receptors are widely expressed in the liver and other steroidogenic organs like adrenals and gonads and bring about selective uptake of cholesteryl esters from HDL particles (6). Apart from binding to HDL particles, they bind to several other ligands like low-density lipoprotein, modified low-density lipoprotein, apoptotic cells, and anionic phospholipids and elicit multiple physiological functions, other than HDL-C metabolism. Some of them are female reproduction, red cell maturation, steroid hormone synthesis, absorption of cholesterol, and other products of lipid digestion through intestinal membranes. Based on these properties, they are aptly classified as multiligand receptors with multiple physiological functions (moonlighting effect of protein) (5).
In liver, these receptors bring about the selective uptake of cholesteryl esters from HDL particles without internalizing the particles, thereby completing the final step in RCT (6). Over-expression of these receptors is known to afford protection against atherosclerosis in rodent models (5, 7). Significantly, in rodents, the expression of these receptors is regulated by several intrinsic factors like hormones [estrogen, testosterone, human chorionic gonadotrophin) and cytokines [tumor necrosis factor (TNF) and interleukin (IL)-6] (8, 9, 10, 11) and extrinsic factors (nutrients like vitamin E and polyunsaturated fatty acids) (12, 13) and infection (14).
The WNIN/Ob rat strain developed from the 80-year-old, Wistar-inbred rat stock colony of the National Centre for Laboratory Animal Sciences of the National Institute of Nutrition (Hyderabad, India) has three phenotypes, namely lean (+/+), carrier (+/−), and obese (−/−). Rats of obese phenotype are hyperphagic, hyperinsulinemic, hypertriglyceridemic, and hypercholesteremic (15). Most of these physical and biochemical traits are comparable with other obese rodent models (16). Furthermore, these rats are also characterized by hyperleptinemia (A. Vajreswari, N. Harishankar, and N.V. Giridharan, unpublished data). Surprisingly, obese rats of this strain have elevated levels of serum HDL-C levels. Another physiological abnormality, namely high peripheral blood reticulocyte count (reflecting defective red cell maturation), is also observed in the obese rats of the WNIN/Ob strain (S.M. Jeyakumar, A. Vajreswari, and N.V. Giridharan, unpublished data).
Studies on SR-BI−/− null mice have reported elevated serum HDL-C levels in these mice (which is attributed to impaired hepatic clearance of cholesterol). These mice also display a defective erythroid differentiation process (which is reflected by higher peripheral blood reticulocyte count), thereby implicating these receptors in the regulation of HDL-C metabolism through RCT and the red cell maturation process (17). Based on the well-established role of SR-BI receptors in RCT and the observed elevated serum HDL-C levels in the obese rats, it was hypothesized that these obese rats might be having an underexpression of these receptors. To test this hypothesis, SR-BI receptor status in relation to serum HDL-C levels was studied in the obese and lean phenotypes of WNIN/Ob strain and compared. In our previous studies, we have demonstrated significant reduction in body weight gain and adiposity index, especially retroperitoneal adipose tissue mass of obese rats in response to vitamin A supplementation. In the present study, we tested whether vitamin A supplementation has any impact on abnormal HDL-C metabolism of obese rats of the WNIN/Ob strain, possibly by altering SR-B1 receptor expression.
Research Methods and Procedures
Animals and Diet
Male, 7-month-old, 12 lean and 12 obese rats of the WNIN/Ob strain were obtained from the National Centre for Laboratory Animal Sciences and broadly divided into two groups, A and B, each consisting of 12 lean and 12 obese rats, respectively, and each was further divided into two subgroups (A-I and A-II and B-I and B-II) having six rats in each. Subgroups A-I and B-I received the stock diet, which provided 2.6 mg vitamin A/kg diet, whereas subgroups A-II and B-II received a high vitamin A (retinyl palmitate)-containing diet (129 mg vitamin A/kg diet). Study was approved by Institutional Animal Ethical Committee. The animals were maintained on their respective diets for a period of 2 months. Food and water were provided ad libitum. Daily food intake and weekly body weights were recorded.
Rats were housed individually with an ambient temperature 22.0 ± 1 °C, relative humidity of 50% to 60%, 12-/12-hour light/dark cycle and cared for in accordance with the principle of the guide to the care and use of experimental animals, formulated by the Committee for the Purpose of Control and Supervision on Experiments on Animals, government of India. At the end of 2 months, rats were killed after 12 hours of fasting. Blood was collected from the supra orbital sinus by the inner canthus by fine heparinized capillary tube and allowed to stand. The blood samples were centrifuged to separate serum and stored at −80 °C. Livers were excised, weighed, rapidly frozen in liquid nitrogen, and stored at −80 °C until analysis.
Determination of Serum Lipids
Serum cholesterol and HDL-C levels were measured using commercial enzymatic kits according to the manufacturer's instructions (Biosystems S.A., Barcelona, Spain). Liver lipids were extracted by the method of Folch et al. (18), and liver cholesterol was assayed using a commercial enzymatic kit according to the manufacturer's instructions (Biosystems S.A.).
Immunoblot Analysis of SR-BI Protein
The presence and quantity of SR-BI receptors were assessed by Western blot analysis. Briefly, liver tissue (1.0 gram) was homogenized in 10 volumes of buffer [20 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 0.25 M sucrose, and 5% (v/v) protease inhibitor cocktail] and centrifuged (800g) for 10 minutes, and the supernatant was centrifuged for 60 minutes at 100,000g. The resulting pellet was washed with buffer to remove lipids and dissolved in 0.1 M phosphate buffer (pH 7.5). These membranes were used for immunoblotting of SR-BI. A constant amount of membrane protein (80 to 100 μg) was solubilized in Laemmli buffer, separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membrane (GE Healthcare, Little Chalfont, Buckinghamshire, UK) (19). Equal loading of the protein and transfer were ensured by staining the membranes with Ponseau S. The membranes were blocked in phosphate-buffered saline-0.02% Tween-20 containing 5% non-fat dry milk before incubation with 1:1000 diluted rabbit polyclonal antibodies to rat SR-BI (gift from S. Azhar and S. Susan), washed, and then incubated with goat anti-rabbit IgG antibodies (1:20,000) conjugated with alkaline phosphatase. After extensive washing, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) substrate was added; the developed bands were analyzed and quantified by scanning using densitometer with automatic calibration (GS-710 Imaging Densitometer; Bio-Rad, Hercules, CA).
Total cellular RNA was extracted using a modification of the method of Chomczynski and Sacchi (20). The integrity of the isolated RNA was checked using 1% agarose gels stained with ethidium bromide. The RNA was quantified by spectrophotometric absorption at 260 nm.
1.0 μg of RNA was used to synthesize first-strand cDNA. The reverse transcription (RT) reaction was carried out in a volume of 20 μL using 100 units of Molony murine leukemia virus reverse transcriptase RNase H− enzyme (Finnzymes Qy, Espoo, Finland) and incubated at 37 °C for 60 minutes. The enzyme was inactivated by heating at 95 °C for 5 minutes. A control experiment without reverse transcriptase was performed for each sample to verify that the amplification did not come from residual genomic DNA. An aliquot of cDNA was amplified in a 20-μL reaction mixture using the forward (5′-GCA TTC GGA ACA GTG CA-3′; sense, 940 to 956) and reverse (5′-GAA CAG CTT CTG ACA GCA-3′; antisense, 1260 to 1277) (GenBank accession no. NM_031541) primers to yield a 337-base pair (bp) polymerase chain reaction (PCR) product of SR-BI. For the SR-BI mRNA, the PCR conditions were as follows: denaturation at 94 °C for 1 minute, annealing at 56 °C for 45 seconds, and polymerization at 70 °C for 1 minute with DyNAzyme II DNA Polymerase (Finnzymes Qy). After 26 cycles, 5 μL of the PCR products was separated by electrophoresis and visualized by ethidium bromide staining. To ensure the equal concentration of c-DNA in the PCR reaction, β-actin was amplified as an internal control using the forward (5′-GCC TCT GGT CGT ACC A-3′; sense, 429 to 444) and reverse (5′-TCC TTC TGC ATC CTG TCA-3′; antisense, 912 to 929) (GenBank accession no. NM_031144) primers to yield a 500-bp PCR product. The number of cycles for the semiquantitative RT-PCR assays and the reaction temperature conditions were estimated to be optimal to provide a linear relationship between the amount of input template and the amount of PCR product generated over a wide concentration range (0.5 to 10 μg) of total RNA.
Results were expressed as means ± standard error of the mean of six animals from each group. Statistical significance was determined by Student's t test or one-way ANOVA, and p ≤ 0.05 was considered significant.
Effects of Dietary Vitamin A on Serum and Liver Lipids
Stock diet-fed obese rats (B-I) had 2.3- and 3.0-fold high levels of serum cholesterol and HDL-C, respectively, compared with the age- and sex-matched lean counterparts (A-I) (Table 1).
Table 1. Effect of vitamin A on serum and liver lipids
A I, lean rats; A II, high vitamin A-containing diet-fed rats; B I, stock diet-fed obese rats; B II, obese rats. Data represent the means ± SE of six rats from each group.
Significant at p ≤ 0.05 level (by one-way ANOVA). Comparisons were made between normal vitamin A vs. high vitamin A groups of each phenotype.
Chronic challenging with a high dose of vitamin A resulted in a significant reduction of 1.5-fold in serum cholesterol and 2.3-fold in HDL-C levels in obese rats (B-II) compared with their stock diet-fed obese rats (B-I). On the contrary, such effects were not observed in rats of lean phenotype treated with high vitamin A-containing diet (A-II) as compared with their lean counterparts maintained on 2.6 mg of vitamin A-containing diet (A-I) (Table 1). In addition, liver cholesterol levels were not significantly altered by high vitamin A content of the diet both in lean and obese phenotypes (A-II and B-II) when compared with their respective controls (A-I and B-I) (Table 1).
Basal Expression of Hepatic SR-BI Receptor (mRNA and Protein Levels)
RT-PCR analysis of hepatic SR-BI mRNA levels showed a significant reduction in the male obese rats (B-I) compared with their sex- and age-matched lean rats (A-I). Furthermore, in line with the RT-PCR data, there was also an underexpression of hepatic SR-BI protein levels from immunoblot data of obese rats (B-I) when compared with their lean counterparts (A-I) (Figure 1).
Impact of Dietary Vitamin A on Hepatic SR-BI Receptor (mRNA and Protein Levels) Expression
Vitamin A supplementation resulted in over-expression of hepatic SR-BI mRNA levels both in the lean and obese phenotypes (A-II and B-II) as against their respective lean and obese rats receiving normal levels of vitamin A through diet (A-I and B-I). Surprisingly, at the protein level, hepatic SR-BI expression was not significantly altered in vitamin A-supplemented lean rats (A-II) compared with their lean rats fed on stock diet (A-I). On the contrary, obese rats supplemented with high doses of vitamin A (B-II) showed significantly higher expression of hepatic SR-BI at the protein level when compared with obese rats maintained on stock diet with 2.6 mg vitamin A/kg diet (B-I) (Figure 2).
The regulation of lipoprotein metabolism in animal models of obesity is very complex due to altered food intake, excessive storage of fat in liver and other organs, and interaction between nutrients and genes of lipogenesis, lipolysis, and lipoprotein metabolism. The role of various receptors in the metabolism of different classes of lipoproteins is very well recognized. Recently, some novel receptors, namely SR-BI, are implicated in HDL-C metabolism, which bring about selective uptake of cholesterol esters from HDL particle by liver (the final step in RCT) and its subsequent excretion as free cholesterol or bile acids through bile (21). Besides their role in RCT, SR-BI receptors of steroidogenic tissues (adrenals and gonads) help in delivering cholesteryl esters for steroidogenesis, thereby implicating them in several other physiological functions (22). These are also involved in cholesterol efflux from cells of peripheral tissues, maintenance of cholesterol, intestinal absorption of fat, and fat digestion products and play a role in erythroid tissue maturation (17, 23, 24, 25).
Obese rats of WNIN/Ob strain have high serum total cholesterol, which is mainly due to abnormally high levels of serum HDL-C. These observations are in line with an underexpression of hepatic SR-BI receptors (at protein and mRNA levels) in the obese rats. These findings are in agreement with those reported for the SR-BI knockout mice model (26). Furthermore, elevated plasma HDL-C levels are also seen in several other genetically obese mutant rodent models such as ob/ob, db/db, fa/fa, tubby, and lethal yellow mice (27) and also in the LA/NIH corpulent obese rat model (28). Thus, elevated plasma HDL-C levels seem to be more or less a generalized phenomenon associated with rodent obesity. Very recently, the elevated plasma HDL-C levels in Ob/Ob mice were shown to be due to underexpression of hepatic SR-B1, and leptin supplementation has normalized plasma HDL-C levels by up-regulation of hepatic SR-BI receptors (29). Interestingly, supplementation with high doses of dietary vitamin A resulted in reduction in serum HDL-C level (which, in turn, reflected in the reduction of serum total cholesterol) with concomitant up-regulation of hepatic SR-BI expression at both protein and gene levels in obese phenotype. Since there were no differences in hepatic cholesterol contents of lean and obese rats and their counterparts supplemented with high doses of dietary vitamin A, it may be presumed that there was no defect in hepatic cholesterol metabolism.
To our knowledge, this is the first report to demonstrate an underexpression of hepatic SR-BI receptors in a leptin-resistant obese rat model and its transcriptional regulation due to vitamin A supplementation that results in normalization of serum HDL-C levels. Intriguingly, although hepatic SR-BI mRNA levels were comparable in vitamin A-supplemented lean and obese phenotypes, a concomitant increase in hepatic SR-BI receptor protein (by Western blot) was observed only in the latter. Furthermore, the supplementation of vitamin A showed beneficial effects such as reduction in body weight gain and total body adiposity mainly due to retroperitoneal white adipose depot loss without affecting their food intake in the obese phenotype (30). However, vitamin A toxicity symptoms such as reduced food intake, alopecia, partial paralysis of hind limbs, depressed growth, and occasional bleeding from the nose were not observed in these rats during the experimental period, thereby suggesting that vitamin A at the dose given was within the safe limits (15).
Furthermore, vitamin A feeding exacerbated the serum triglyceride levels in both lean and obese phenotypes. However, the effect was significant in obese rats alone (S.M. Jeyakumar, A. Vajreswari, and N.V. Giridharan, unpublished data). Serum retinol (15) and free fatty acid levels were unaltered in either of the phenotypes fed a high vitamin A diet (S.M. Jeyakumar, A. Vajreswari, and N.V. Giridharan, unpublished data). Hypertriglyceridemia is a common phenomenon associated with the feeding of a high dose of vitamin A and has been reported earlier (31). Furthermore, besides exerting anti-obesity effects, nutrients like conjugated linoleic acid (32), polyunsaturated fatty acid-rich diet (S.M. Jeyakumar, A. Vajreswari, and N.V. Giridharan), and one-third food restriction (N. Harishankar and N.V. Giridharan, unpublished data) are also known to induce hypertriglyceridemia in the obese rats of WNIN/Ob strain.
There is rich vein of scientific evidence to suggest that a multitude of intrinsic factors [several hormones like estrogens, testosterone, and human chorionic gonadotrophin hormone and several cytokines like IL-1 and TNFα and an adipokine leptin have a regulatory role on SR-BI expression (8, 9, 10, 11, 29)]. It has also been reported that in general, obesity is also associated with over-expression of some of these cytokines (33). TNFα over-expression in obesity and its role as a molecular link between obesity and insulin resistance is reported in obese rodent models (34). Furthermore, TNFα and IL-1 negatively regulate SR-BI expression (10). Although the specific role of leptin in relation to HDL metabolism and hepatic SR-B1 receptor regulation has been established in leptin-deficient ob/ob mice (29), it may not be of much relevance in the context of current leptin-resistant/hyperleptinemic obese rats of the WNIN/Ob strain. Therefore, the observed underexpression of hepatic SR-BI in the obese rat could be due to elevated levels of some of these cytokines, which needs further exploration.
Among the nutrients that regulate SR-B1 expression, vitamin E and polyunsaturated fatty acid are studied in detail (12, 13). In a very systematic study, Witt et al. have established an inverse relationship between plasma and tissue levels and hepatic expression of SR-B1 (12). Based on these results, it has been postulated that vitamin E status plays a crucial role in the regulation of hepatic SR-B1 expression. Interestingly, though, vitamin E status altered SR-B1 receptor protein, there is no difference in SR-B1 gene expression levels, thereby implicating the role of certain posttranscriptional modifications/mechanisms in bringing about differential expression of SR-B1 receptor (protein) as a function of vitamin E status (12). As discussed earlier, the basal SR-BI protein levels in stock diet-fed lean and obese rats, and vitamin A-supplemented obese rats correlated very well with the gene expression (mRNA) levels. On the other hand, in vitamin A-treated lean rats, SR-BI protein levels remained unaltered, despite higher mRNA levels. Thus, these results suggest the role of different posttranscriptional events in the regulation of SR-BI receptor levels in lean and obese phenotypes of the WNIN/Ob strain in response to vitamin A treatment. Interestingly, these observations are in line with those reported for vitamin E effects on the expression of SR-BI, cell adhesion molecules, and ILs (12). Certain posttranscriptional modifications have been implicated to explain the phenomenon of SR-BI up-regulation in apolipoprotein-E knockout mice (35), down-regulation of SR-BI by vitamin E supplementation to vitamin E-depleted rats (12). In addition, the down-regulation of SR-BI by bacterial lipopolysaccharides in human macrophages has been attributed to modification and subsequent destabilization of mRNA (36). Indirect evidence of the posttranscriptional regulation of SR-BI is also reported in several organs in rat, including liver (6).
The results of the present study clearly suggest an underexpression of these receptors in the obese phenotype, which could be either due to overexpression of certain cytokines or lower hepatic vitamin A status (as shown in vitamin E-depleted rat study) or both. Although data on basal expression of cytokines are not available, data on vitamin A status suggest that lean rats maintained on stock diet had higher levels of hepatic vitamin A compared with their obese counterparts (15). Furthermore, vitamin A supplementation, although it resulted in elevated hepatic stores of vitamin A in obese rats, these levels were considerably lower than those observed in lean rats receiving identical dietary treatment.
Interestingly, another physiological abnormality related to SR-BI underexpression, namely elevated reticulocyte count (impaired erythroid differentiation), was also observed in obese rats, which was also normalized by chronic vitamin A supplementation to obese rats (S.M. Jeyakumar, A. Vajreswari, and N.V. Giridharan, unpublished data). However, no such abnormality was observed in stock diet-fed lean rats with normal SR-BI expression. Importantly, chronic dietary vitamin A supplementation, although it significantly raised hepatic vitamin A status and SR-BI mRNA levels, had no effect on SR-BI protein expression, serum HDL-C levels, and reticulocyte count in lean rats.
In view of the anti-atherogenic and cardioprotective roles of SR-BI and extensive structural and functional homology between rodent and human SR-BI, the current observation that the leptin-resistant obese rats have an underexpression of these receptors establishes them as the “physiological SR-BI knock-down model” and reconfirms their role in HDL-C metabolism. Interestingly, a simple, single micronutrient (vitamin A) supplementation at high but non-toxic doses could effectively correct the physiological abnormalities like abnormal serum HDL-C levels and reticulocyte count (S.M. Jeyakumar, A. Vajreswari, and N.V. Giridharan, unpublished data) in a genetically obese rat model by up-regulating SR-BI receptor expression, which underscores the importance of nutrient–gene interactions in health and disease conditions. Therapeutic and pharmacological potential of vitamin A supplementation in correcting human obesity-associated abnormal lipoprotein metabolism should be exploited, after establishing the minimum effective dose of vitamin A and the mechanism through which vitamin A regulates SR-BI expression in this obese rat model.
We gratefully acknowledge Salman Azhar and Sucheta Susan (Stanford University School of Medicine, Veterans Administration Palo Alto Health Care System, Palo Alto, CA) for the generous gift of SR-BI antibodies. S.M. Jeyakumar thanks the Indian Council of Medical Research, India for the award of a research fellowship to carry out this work.